The present invention relates to new viral vectors, to their preparation and to their use in gene therapy. It also relates to pharmaceutical compositions containing said viral vectors. More especially, the present invention relates to recombinant adenoviruses as vectors for gene therapy.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introducing genetic information into the cell or organ affected. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, including various techniques of transfection involving complexes of DNA and DEAE-dextran (Pagano et al., J.Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375) and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, EMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
Among these viruses, the adenoviruses display certain properties which are advantageous for use in gene therapy. In particular, they have a fairly broad host range, are capable of infecting resting cells, do not integrate in the genome of the infected cell and have not been associated to date with pathologies of importance in man. Adenoviruses have thus been used to transfer genes of interest to muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), and the like.
Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome com prises, in particular, an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes (see FIG. 1). The main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region (E1a and E1b in particular) are necessary for viral replication. The E4 and L5 regions, for example, are, for their part, involved in viral propagation. The main late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been sequenced completely, and is accessible on a database (see, in particular, Genebank M73260). Likewise, some portions, or even the whole, of the genome of adenoviruses of different serotypes (Ad2, Ad7, Ad12, and the like) have also been sequenced.
In view of the properties of the adenoviruses mentioned above, the latter have already been used for the transfer of genes in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes (xcex2-gal, OTC, xcex11-AT, cyto-kines, and the like). In each of these constructions, the adenovirus has been modified so as to make it incapable of replication in the infected cell. Thus, the constructions described in the prior art are adenoviruses from which the E1 (E1a and/or E1b) and possibly E3 regions have been deleted, in which regions, the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Other constructions contain a deletion in the E1 region and of a non-essential portion of the E4 region (WO94/12649). Nevertheless, the vectors described in the prior art have some drawbacks which limit their use in gene therapy. In particular, the batches of recombinant viruses of the type described in the prior art may be contaminated with replicative particles, in particular of the wild type.
At the present time, the vectors derived from adenoviruses are, in effect, produced in a complementation line (line 293) in which a portion of the adenovirus genome has been integrated. More specifically, line 293 contains the left-hand end (approximately 11-12%) of the adenovirus serotype 5 (Ad5) genome, comprising the left-hand ITR, the encapsidation region and the E1 region, including E1a, E1b and a portion of the region coding for the pIX protein. This line is capable of trans-complementing recombinant adenoviruses which are defective for the E1 region, that is to say lacking all or part of the E1 region, necessary for replication. In effect, E1xe2x88x92 recombinant adenoviruses may be prepared in 293 cells as a result of the good trans-complementation of the E1 region contained in this line. Nevertheless, there are zones of homology between the adenovirus region integrated in the genome of the line and the DNA of the recombinant virus which it is desired to produce. As a result, during production, different recombination events may take place, generating replicative viral particles, in particular adenoviruses of the E1+ type. As shown in FIG. 2, the outcome can be a single recombination event followed by chromosome breakage (FIG. 2A), or a double recombination (FIG. 2B). These two types of modification lead to a replacement of the left-hand portion of the recombinant DNA, lacking a functional E1 region, by the corresponding portion present in the genome of the cell, which carries a functional copy of the E1 region. Moreover, in view of the high titres of recombinant vector produced by line 293 (greater than 1012), the probability of these recombination events taking place is high. In fact, it has been found that many batches of defective recombinant adenoviral vectors were contaminated with replicative viral particles.
Contamination with replicative particles constitutes a major drawback. In effect, the presence of such particles in therapeutic compositions would induce in vivo a viral propagation and an uncontrolled dissemination, with risks of an inflammatory reaction, of recombination, and the like. Hence the contaminated batches cannot be used in human therapy.
The present invention enables these drawbacks to be remedied. The present invention describes, in effect, new constructions permitting the production of defective recombinant adenoviruses completely lacking contamination with replicative particles. The present invention also describes a method for the production of these recombinant adenoviruses. It thus provides new defective recombinant vectors derived from adenoviruses which are especially suitable for use in gene therapy, in particular for the transfer and expression of genes in vivo.
The present invention lies more especially in the construction of defective recombinant adenoviruses comprising an adenovirus genome whose genetic organization is modified, and the possible recombination of which with the genome of the producing line leads to the generation of non-replicative and/or non-viable viral particles. The Applicant has now shown that it is possible to modify the genomic organization of the adenovirus in order to avoid the production of replicative particles during the production of the stocks.
A first subject of the present invention hence relates to a recombinant adenovirus comprising an adenovirus genome (i) whose E1 region is inactivated, (ii) whose genomic organization is modified, and (iii) the possible recombination of which with the genome of the producing line leads to the generation of non-viable viral particles.
For the purposes of the present invention, genetic or genomic organization is understood to mean the arrangement of the different genes or functional regions present in the genome of the wild-type adenovirus as shown in FIG. 1. A modified genetic or genomic organization hence corresponds to a genome in which some genes or some regions are not in their original position. Thus, some genes or some regions may be moved from the genome and inserted at another site. It is also possible to insert a given gene or region at a particular site, and to eliminate or inactivate the original region (by mutation, deletion, insertion, and the like).
The term non-viable viral particle denotes, for the purposes of the invention, an adenovirus incapable of replicating its DNA and/or of propagating autonomously in the infected cells. A non-viable viral particle hence possesses an adenovirus genome lacking at least sequences necessary for its replication and/or its propagation in the infected cell. These regions may be either removed (wholly or partially), or rendered non-functional, or substituted by other sequences. The sequences necessary for replication and/or propagation are, for example, the E1 region, the E4 region or the L5 region. More especially, as regards the E4 region, the important genes are the ORF3 and ORF6 genes.
The Applicant has shown, more especially, that it is possible to move a function essential to viral replication or propagation without affecting the properties of the adenovirus as a vector for gene therapy, namely its high power of infection of cells, in particular human cells, and its capacity to transfer a gene of interest effectively to said cells. Thus, in a preferred embodiment, the subject of the present invention is recombinant adenoviruses in which a region essential to viral replication and/or propagation is present in a genomic position other than its original position. Advantageously, this region lies in or in proximity to another genomic region which is rendered non-functional.
The vectors of the invention are especially advantageous, since they enable large genes of interest to be incorporated and can be produced at high titres without the production of any contaminating replicative viral particle.
In the vectors of the invention the E1 region or any other region may be inactivated or rendered nonfunctional by different techniques known to a person skilled in the art, and in particular by elimination, substitution, deletion and/or addition of one or more bases. Such modifications may be obtained in vitro (on isolated DNA) or in situ, for example by means of genetic engineering techniques or alternatively by treatment by means of mutagenic agents. Said genetic modification or modifications may be localized in a coding portion of the region or outside a coding region, and for example in the regions responsible for the expression and/or transcriptional regulation of said genes. The inactivation may hence manifest itself in the production of proteins which are inactive as a result of structural or conformational modifications, by the absence of production, by the production of proteins having impaired activity or alternatively by the production of natural proteins at a level which is attenuated or according to a desired mode of regulation.
Among the mutagenic agents which can be used for the inactivation, there may be mentioned, for example, physical agents such as energetic radiation (X, xcex3, ultraviolet rays, and the like), or chemical agents capable of reacting with different functional groups of the DNA bases, and for example alkylating agents [ethyl methanesulphonate (EMS), N-methyl-Nxe2x80x2-nitro-N-nitroso-guanidine, N-nitroquinoline 1-oxide (NQO)], bialkylating agents, intercalating agents, and the like.
The genetic modifications may also be obtained by gene disruption, for example according to the protocol initially described by Rothstein [Meth. Enzymol. 101 (1983) 202]. In this case, all or part of the coding sequence is preferably disrupted to permit the replacement, by homologous recombination, of the genomic sequence by a non-functional or mutant sequence.
Preferably, in the adenoviruses of the invention, the region in question is inactivated by mutation and/or deletion of one or more bases. Still more preferably, it is inactivated by total or partial deletion.
More especially, deletion is understood to mean any elimination of all or part of the gene in question. This can apply, in particular, to all or part of the coding region of said gene, and/or of all or part of the transcription promoter region of said gene. The elimination may be performed by digestion by means of suitable restriction enzymes, followed by ligation, according to the standard techniques of molecular biology, as illustrated in the examples.
As a special preference, the inactivation of the genes is carried out in such a way that it affects only the gene in question and not the other viral genes, in particular the neighbouring genes. Moreover, since some modifications such as point mutations are inherently capable of being corrected or attenuated by cellular mechanisms, it is especially preferable for the inactivation to be completely segregationally stable and/or irreversible.
In the case of inactivation by total or partial deletion, the region essential to viral viability preferably lies in or in proximity to the deletion site. It is, however, possible to use other insertion sites, such as, for example, restriction sites already present in the wild-type genome. In this connection, it is nevertheless preferable for the insertion to be carried out at least in proximity to the deletion site, that is to say outside the deletion site, but sufficiently ready for recombination events not to be able to take place in the space separating the deletion site and the insertion site. Preferably, the distance between the deletion site and the insertion site should not exceed 50 bp.
In a preferred embodiment of the present invention, the region essential to viral replication and/or propagation is moved so as to be included in the inactivated E1 region and/or the E3 region.
According to an especially advantageous embodiment, in the recombinant adenoviruses of the present invention, the E1 region is inactivated by deletion of a PvuII-BglII fragment extending from nucleotide 454 to nucleotide 3328 on the Ad5 adenovirus sequence. This sequence is accessible in the literature and also on a database (see, in particular, Genebank No. M73260). In another preferred embodiment, the E1 region is inactivated by deletion of a HinfII-Sau3A fragment extending from nucleotide 382 to nucleotide 3446.
The region essential to viral replication and/or propagation according to the present invention is advantageously chosen from all or part of the E4 region and/or of the pIX-IVa2 region and/or of the L5 region, and the like.
In an especially preferred embodiment of the present invention, the essential region consists of all or a functional portion of the E4 region, and it is inserted in or in proximity to the E1 deletion site. According to this embodiment, the E4 region, essential to viral propagation, is inserted in a position other than its original position, so that this region is absent in any construction which might result from recombination with the genome of the producing line (see FIG. 3). Hence the subject of the present invention is also a recombinant adenovirus whose genome is distinguished by the presence of inactivated E1 and E4 regions, and in which all or a functional portion of the E4 region is inserted in or in proximity to the E1 region.
An adenoviral vector of this kind preferably comprises two ITRs, an encapsidation region, a deletion in the E1 region in which all or a functional portion of E4 is inserted, and an inactivated original E4 region.
The E4 region is involved in regulation of the expression of the late genes, in the stability of the late nuclear RNAs, in abolition of the expression of the proteins of the host cell and in the efficacy of the replication of the viral DNA. Mutants lacking E4 are incapable of propagating. E4 thus constitutes a region essential to viral replication and/or propagation. This E4 region consists of 7 open reading frames, designated ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7 (FIG. 4). Among these, ORF3 and ORF6 are the two genes essential to viral propagation. Each of these genes is capable of inducing viral propagation. On the other hand, inactivation of the E4 region involves the inactivation of ORF3 and ORF6.
In a particular embodiment, in the vectors of the invention, the whole of the E4 region is inserted in or in proximity to the E1 deletion site. The region can correspond, in particular, to an MaeII-MscI fragment corresponding to nucleotides 35835-32720.
In another particular embodiment, only a functional portion of E4, that is to say a portion sufficient to permit viral propagation, is inserted. This portion comprises at least one functional ORF3 or ORF6 gene. Preferably, the functional portion of E4 consists essentially of ORF3 or ORF6. As an example, these coding frames may be isolated from the E4 region in the form of PvuII-AluI and BglII-PvuII fragments, respectively, corresponding to nucleotides 34801-34329 and 34115-33126, respectively.
Advantageously, the E4 region or the functional portion of this region also comprises a transcription promoter region. The promoter in question can be that of the E4 region or any other functional promoter, such as viral (E1a, SV40, RSV LTR, and the like), eukaryotic or mammalian promoters. Preferably, the promoter used is the promoter of the E4 region.
As mentioned above, the functional portion of E4 inserted in E1 does not necessarily correspond to the portion of E4 deleted in the original position. Thus, the initial region may be inactivated by point mutation (without deletion), and a functional E4 region inserted in E1. Likewise, the initial E4 region may be completely deleted, and only a functional portion inserted in E1.
Inactivation of the E4 region implies, for the purposes of the invention, the functional inactivation of at least the ORF3 and ORF6 regions. These original regions may be inactivated by any technique known to a person skilled in the art. In particular, all the methods given above may be applied to the inactivation of ORF3 and ORF6 or any additional region of E4. As an example, deletion of the E4 region of the virus Ad2 dl808 or of the viruses Ad5 dl1004, Ad5 dl1007, Ad5 dl1011 or Ad5 dl1014 may be used in the context of the invention (see Example 3).
These adenoviruses may be obtained, for example, by cotransfection into a producing line of a first plasmid carrying the left-hand portion of the genome of the virus which it is desired to produce (possessing a deletion in the E1 region in or in proximity to which at least a functional portion of E4 is inserted), and a viral genomic DNA fragment supplying the right-hand portion of the genome of the virus (possessing an inactivated E4 region). After recombination, the viruses produced are amplified and isolated. These adenoviruses may also be obtained by interchanging the ends of the genome, comprising the ITRs plus the adjacent region. In this connection, the subject of the invention is also a recombinant adenovirus whose genome possesses an inactivated E1 region, and in which the left-hand end, comprising the ITR and the encapsidation region, and the right-hand end, comprising the ITR and all or a functional portion of the E4 region, are interchanged. More especially, the left-hand end comprising the left-hand ITR and the encapsidation region is contained in the first 382 nucleotides of the Ad5 adenovirus genome (for example up to the HinfI site). Likewise, the right-hand end comprising the right-hand ITR and all or a functional portion of the E4 region, including the promoter of the E4 region, is contained in the last 3215 nucleotides of the Ad5 adenovirus genome (for example from the MscI site at position 32720). The techniques of a person skilled in the art enable a recombinant virus according to the invention to be constructed in which the right-hand ITR and all or part of the E4 region are now located on the left-hand side of the virus, followed by the region 3446-32720 of the Ad5 adenovirus genome, then the encapsidation sequence and the left-hand ITR which now becomes the right-hand end of the recombinant virus (see FIG. 5). The genome of the recombinant adenovirus thereby obtained is especially advantageous, since the essential E4 region moved to the left is maintained in its natural environment and hence under optimal conditions of activity for a high-titre infectious cycle. Furthermore, this region now precedes the region whose presence in the genome of the producing line 293 was the source of the appearance of viable particles.
In another, most especially preferred embodiment of the present invention, the essential region consists of the region coding for the pIX and IVa2 proteins, and it is inserted in the E3 region, optionally as a replacement for deleted sequences (see FIG. 6). More especially, the region coding for the pIX and IVa2 proteins is included in a BglII-NruI fragment corresponding to nucleotides 3328 to 6316 on the wild-type Ad5 adenovirus sequence. In this embodiment, the possible recombination of the recombinant adenovirus with the adenovirus region integrated in the producing line generates only non-viable viral particles, since the main late genes essential to viability have been deleted from them.
According to a particular embodiment of the invention, two essential regions of the adenovirus genome are moved from their original positions. More preferably, these essential regions are represented by the region coding for the pIX protein and the region coding for all or only a functional portion of the E4 region. According to a preferred embodiment, they will be moved to the E1 region, as a replacement for deleted sequences and preserving or otherwise the orientation of their reading frame.
By way of illustration of this type of construction, reference may be made more especially to the construction shown in FIG. 8. In this construction, the region coding for the pIX protein is moved into the deleted E1 region, on the right-hand side of the left-hand ITR which has become, right-hand end of the recombinant virus. The region coding for the pIX protein is, in addition, placed therein reading in the reverse direction. As regards the essential region of E4, this is represented therein by the ORF3-ORF6/7 genes under the control of the E4 promoter, and is also inserted therein in the E1 deletion site, between the region coding for the pIX protein and the region coding for the IVa2 protein, whose position has not been affected; in the case of the specific construction of FIG. 8, the two regions coding for the pIX protein and the IVa2 protein, respectively, possess separate polyadenylation sites.
A construction of this kind is especially advantageous from the standpoint of reliability and safety. In effect, any spurious recombination between 2 viral molecules of this type, in the E4 region for example, will lead to a recombinant virus bereft of its encapsidation sequence. Likewise, a recombination between a viral molecule of this kind with the complementary region of said adenovirus, the region being integrated in a producing cell line, will generate only viral particles from which their main late genes essential to their viability have been deleted.
As mentioned above, the recombinant adenoviruses according to the invention advantageously comprise the ITR sequences and a region permitting encapsidation.
The inverted repeat sequences (ITR) constitute the origin of replication of the adenoviruses. They are localized at the 3xe2x80x2 and 5xe2x80x2 ends of the viral genome (see FIG. 1), from where they may be readily isolated according to the standard techniques of molecular biology known to a person skilled in the art. The nucleotide sequence of the ITR sequences of human adenoviruses (especially of the serotypes Ad2 and Ad5) is described in the literature, as well as those of canine adenoviruses (in particular CAV1 and CAV2). As regards the Ad5 adenovirus for example, the left-hand ITR sequence corresponds to the region comprising nucleotides 1 to 103 of the genome.
The encapsidation sequence (also designated Psi sequence) is needed for encapsidation of the viral genome. This region must hence be present in order to permit the preparation of defective recombinant adenoviruses according to the invention. The encapsidation sequence is localized in the genome of the wild-type adenoviruses, between the left-hand ITR and the E1 gene (see FIG. 1). In the adenoviruses of the invention, it may be localized next to either the left-hand ITR or the right-hand ITR (see FIG. 5). It may be isolated or synthesized artificially by the standard techniques of molecular biology. The nucleotide sequence of the encapsidation sequence of human adenoviruses (especially of the serotypes Ad2 and Ads) is described in the literature, as well as those of canine adenoviruses (in particular CAV1 and CAV2). As regards the Ad5 adenovirus for example, a functional encapsidation sequence lies between nucleotides 194 and 358 of the genome.
Moreover, the adenoviruses according to the invention can possess other modifications in respect of their genome. In particular, other region may be deleted in order to increase the capacity of the virus and to reduce these side effects associated with the expression of viral genes. Thus, all or part of the E3 region in particular may be deleted.
Recombinant adenoviruses according to the invention possess especially attractive properties for use in gene therapy. These vectors combine, in effect, very high infection, safety and gene transfer capacity properties.
Advantageously, the recombinant adenoviruses of the invention contain, in addition, a heterologous nucleic acid sequence whose transfer to a cell, organ or organism and/or expression therein is/are sought.
In particular, the heterologous DNA sequence can contain one or more therapeutic genes. The therapeutic genes which can thus be transferred are any gene whose transcription and, where appropriate, translation in the target cell generate products having a therapeutic effect.
The genes in question can, in particular, be ones coding for proteinaceous products having a therapeutic effect. The proteinaceous product thus encoded can be a protein, a peptide, an amino acid, and the like. This proteinaceous product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter does not display any pathology). In this case, the expression of a protein makes it possible, for example, to compensate for an insufficient expression in the cell or for the expression of a protein that is inactive or poorly active as a result of a modification, or alternatively to overexpress said protein. The therapeutic gene can also code for a mutant of a cellular protein, having enhanced stability, modified activity, and the like. The proteinaceous product can also be heterologous with respect to the target cell. In this case, an expressed protein can, for example, supplement or supply an activity which is deficient in the cell, enabling it to combat a pathology.
Among the products which are therapeutic for the purposes of the present invention, there may be mentioned, more especially, enzymes, blood derivatives, hormones, lymphokines, namely interleukins, interferons, TNF, and the like (FR 92/03120), growth factors, neuro-transmitters or their precursors or synthetic enzymes, trophic factors, namely BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, and the like; apolipoproteins, namely ApoAI, ApoAIV, ApoE, and the like (FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), tumor-suppressing genes, namely p53, Rb, Rap1A, DCC, k-rev, and the like (FR 93/04745), genes coding for factors involved in coagulation, namely factors VII, VIII, IX, and the like, suicide genes, namely those for thymidine kinase, cytosine desaminase, and the like; or alternatively all or part of a natural or artificial immunoglobulin (Fab, ScFv, and the like), and the like.
The therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell enables the expression of cellular genes or the transcription of cellular mRNA to be controlled. Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs, and can thus block their translation into protein, according to the technique described in Patent EP 140,308.
The therapeutic gene can also be a gene coding for an antigenic peptide capable of generating an immune response in humans. In this particular embodiment, the invention hence makes it possible to produce vaccines enabling humans to be immunized, in particular against microorganisms or viruses. Such antigenic peptides can be, in particular, ones specific to the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185,573) or the pseudorabies virus, or alternatively tumor-specific (EP 259,212).
Generally, the heterologous nucleic acid sequence also comprises a transcription promoter region which is functional in the infected cell, as well as a region located on the 3xe2x80x2 side of the gene of interest, and which specifies a transcription termination signal and a polyadenylation site. This set of elements constitutes the expression cassette. As regards the promoter region, this can be a promoter region which is naturally responsible for the expression of the gene in question when the region is capable of functioning in the infected cell. The promoter regions can also be ones of different origin (responsible for the expression of other proteins, or even synthetic regions). In particular, they can be promoter sequences of eukaryotic or viral genes. For example, they can be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they can be promoter sequences originating from the genome of a virus, including the adenovirus used. In this connection, the promoters of the E1A, MLP (major late promoter), CMV (cytomegalovirus), RSV (Rous sarcoma virus), and the like, genes may be mentioned for example. In addition, these promoter regions may be modified by the addition of activator or regulatory sequences, or sequences permitting a tissue-specific or -preponderant expression. Moreover, when the heterologous nucleic acid does not contain promoter sequences, it may be inserted into the genome of the defective virus downstream of such a sequence.
Moreover, the heterologous nucleic acid sequence can also contain, especially upstream of the therapeutic gene, a signal sequence directing the synthesized therapeutic product into the pathways of secretion of the target cell. This signal sequence can be the natural signal sequence of the therapeutic product, but it can also be any other functional signal sequence, or an artificial signal sequence.
The cassette for expression of the therapeutic gene may be inserted at different sites of the genome of the recombinant adenovirus according to the invention. It may, in the first place, be inserted at the site of the E1 deletion. In this case, it is localized next to (on the 5xe2x80x2 or 3xe2x80x2 side) the region or the functional portion of E4. It may also be inserted in the E3 region, in addition to or as a substitute for sequences. It may also be localized in the inactivated E4 region.
Still in an especially advantageous embodiment, the vectors of the invention possess, in addition, a functional E3 gene under the control of a heterologous promoter. More preferably, the vectors possess a portion of the E3 gene permitting expression of the gp19K protein. This protein makes it possible, in effect, to prevent the adenovirus vector from becoming the subject of an immune reaction which (i) would limit its action and (ii) might have undesirable side effects.
The recombinant adenoviruses according to the invention may be of diverse origins. There are, in effect, different serotypes of adenovirus, the structure and properties of which vary somewhat but which display a comparable genetic organization. As a result, the teachings described in the present application may be readily reproduced by a person skilled in the art for any type of adenovirus.
More especially, the adenoviruses of the invention may be of human, animal or mixed (human and animal) origin.
As regards adenoviruses of human origin, it is preferable to use those classified in group C. More preferably, among the different serotypes of human adenovirus, it is preferable to use adenoviruses type 2 or 5 (Ad2 or Ad5) in the context of the present invention.
As mentioned above, the adenoviruses of the invention may also be of animal origin, or may contain sequences originating from adenoviruses of animal origin. The Applicant has, in effect, shown that adenoviruses of animal origin are capable of infecting human cells with great efficacy, and that they are incapable of propagating in the human cells in which they have been tested (see Application FR 93/05954). The Applicant has also shown that adenoviruses of animal origin are in no way trans-complemented by adenoviruses of human origin, thereby eliminating any risk of recombination and propagation in vivo in the presence of a human adenovirus, which can lead to the formation of an infectious particle. The use of adenoviruses or of regions of adenoviruses of animal origin is hence especially advantageous, since the risks inherent in the use of viruses as vectors in gene therapy are even lower.
The adenoviruses of animal origin which may be used in the context of the present invention can be of canine, bovine, murine (for example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian (for example: SAV) origin. More especially, among avian adenoviruses, there may be mentioned the serotypes 1 to 10 which are available in the ATCC, such as, for example, the strains Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), K-11 (ATCC VR-921) or alternatively the strains referenced ATCC VR-831 to 835. Among bovine adenoviruses, the different known serotypes may be used, and in particular those available in the ATCC (types 1 to 8) under the references ATCC VR-313, 314, 639-642, 768 and 769. There may also be mentioned murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-528), ovine adenovirus type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340), porcine adenovirus 5359, or simian adenoviruses such as, in particular, the adenoviruses referenced in the ATCC under the numbers VR-591-594, 941-943, 195-203, and the like.
Among the different adenoviruses of animal origin, it is preferable in the context of the invention to use adenoviruses or regions of adenoviruses of canine origin, and in particular all strains of CAV2 adenoviruses [strain Manhattan or A26/61 (ATCC VR-800) for example]. Canine adenoviruses have been subjected to many structural studies. Thus, complete restriction maps of CAV1 and CAV2 adenoviruses have been described in the prior art (Spibey et al., J. Gen. Virol. 70 (1989) 165), and the E1a and E3 genes as well as the ITR sequences have been cloned and sequenced (see, in particular, Spibey et al., Virus Res. 14 (1989) 241; Linnxc3xa9, Virus Res. 23 (1992) 119, WO 91/11525).
The defective recombinant adenoviruses according to the invention may be prepared in different ways.
A first method consists in transfecting the DNA of the (defective) recombinant virus prepared in vitro into a competent cell line, that is to say one carrying in trans all the functions necessary for complementation of the defective virus. These functions are preferably integrated in the genome of the cell, thereby reducing the risks of recombination and endowing the cell line with enhanced stability. In the case of adenoviruses in which only the E1 region is deficient, the preferred line is line 293.
A second approach consists in cotransfecting the DNA of the defective recombinant virus prepared in vitro and the DNA of one or more helper viruses or plasmid into a suitable cell line. According to this method, it is not necessary to have at one""s disposal a competent cell line capable of complementing all the defective functions of the recombinant adenovirus. A part of these functions is, in effect, complemented by the helper virus or viruses. This/these helper virus (es) are themselves defective. The preparation of defective recombinant adenoviruses of the invention according to this method is also illustrated in the examples.
In this connection, the present application also describes construction of plasmids carrying the modified left-hand portion of the Ads adenovirus genome (plasmids of the pCO1-E4 series, for example). These plasmids are especially useful for the construction of recombinant adenoviruses as vectors for gene therapy. Thus, the pCO1-E4 plasmids carry the left-hand region of the adenovirus genome, from the left-hand ITR to nucleotide 6316, with a deletion of the region lying between nucleotides 382 and 3446, corresponding to the E1 locus, in which region all or a functional portion of E4 is inserted. The pCO1-E4 plasmids contain, moreover, a multiple cloning site permitting the incorporation of a heterologous nucleic acid sequence of interest. The pCO1-E4 plasmids may be used to prepare the defective recombinant adenovirus by cotransfection with a DNA, preferably of viral origin, corresponding to the right-hand portion of the genome of the adenovirus possessing an inactivated E4 region, in a competent cell line. As regards the latter DNA, it may originate from the genome of a defective virus such as Ad2 dl808 from which the E4 region has been deleted (Weinberg et al., J. Virol. 57 (1986) 833), Ad5 dl1004, Ad5 dl1007, Ad5 dl1011 or Ad5 dl1014, and the like (see examples). In this connection, the invention also relates to a method for preparing recombinant adenoviruses lacking replicative particles, according to which a competent cell line is cotransfected with
a first DNA comprising the left-hand portion of the genome of said adenovirus, possessing a deletion in the E1 region in or in proximity to which at least a functional portion of the E4 region is inserted, and
a second DNA comprising at least the right-hand portion of the genome of said adenovirus, possessing an inactivated E4 region, and a portion of adenovirus in common with the first DNA,
and the adenoviruses produced by homologous recombination between said DNAs are recovered.
Among the cell lines which can be used, the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) may be mentioned in particular. As stated above, this line contains, in particular, integrated in its genome, the left-hand portion of the Ad5 human adenovirus genome (12%). Advantageously, in the method of the invention, the first DNA is chosen from plasmids of the pCO1-E4 type.
Moreover, in order to prepare a recombinant adenovirus comprising a therapeutic gene, the first or the second DNA employed in the method of the invention carries, in addition, a heterologous DNA sequence of interest.
Thereafter, the recombinant viruses which have multiplied are recovered, purified and amplified according to the standard techniques of virology.
The pCO1-E4 plasmids thus enable recombinant adenoviruses to be constructed carrying a deletion in the E1 region extending from nucleotide 382 to nucleotide 3446, in which region all or a functional portion of E4 is inserted, as well as, where appropriate, a therapeutic gene.
The present invention also relates to any pharmaceutical composition comprising one or more defective recombinant adenoviruses as are described above. The pharmaceutical compositions of the invention may be formulated with a view to topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, and the like, administration.
Preferably, the pharmaceutical composition contains vehicles which are pharmaceutically acceptable for an injectable formulation. These can be, in particular, sterile, isotonic saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, and the like, or mixtures of such salts), or dry, in particular lyophilized, compositions which, on adding sterilized water or physiological saline, as the case may be, enable injectable solutions to be formed.
The doses of virus used for the injection may be adapted in accordance with different parameters, and in particular in accordance with the mode of administration used, the pathology in question, the gene to be expressed or the desired period of treatment. Generally speaking, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu, and preferably 106 to 1010 pfu. The term pfu (plaque forming unit) corresponds to the infectious power of a solution of virus, and is determined by infecting a suitable cell culture and measuring, generally after 15 days, the number of plaques of infected cells. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.
Depending on the heterologous DNA sequence inserted, the adenoviruses of the invention may be used for the treatment or prevention of many pathologies, including genetic diseases (dystrophy, cystic fibrosis, and the like), neurodegenerative diseases (Alzheimer""s, Parkinson""s, ALS, and the like), cancers, pathologies associated with disorders of coagulation or with dyslipo-proteinaemias, pathologies associated with viral infections (hepatitis, AIDS, and the like), and the like.
The present invention will be described more completely by means of the examples which follow, which should be considered to be illustrative and non-limiting.